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2.1 Mice

All animal work was conducted in accordance with Institutional Animal Care and Use Guidelines of Columbia University. Mice were housed in specific-pathogen-free conditions. C57BL/6 mice, Pax5-IRES-hCD2 reporter mice (Fuxa & Busslinger, 2007), P14 TCR transgenic (P14) mice recognizing LCMV peptide gp33-41/Db, C57BL/6 OTII mice expressing an I-Ab- restricted TCR specific for ovalbumin amino acids 323-339, AMPKα1 knockout mice

(Jorgensen et al., 2004) (Prkaa1tm1Vio), and IRF4 knockout mice (Klein et al., 2006) have been previously described. 8-12 week old mice were used for most experiments. Myc-tagged-Glut1 mice were from Dr. Jeffery Rathmell (Young et al., 2011), whereas Glut1fl/fl mice were from Dr. Gerard Karsenty (Wei et al., 2015).

2.2 Cell culture and lymphocyte activation

Spleens from mice were isolated and processed using established protocols. B cells and CD8+ T cells were enriched from spleens by magnetic bead negative selection with the MACS B cell isolation kit (Miltenyi) and the MACS CD8+ T cell isolation kit (Miltenyi), respectively. B cells (5 x 105 cells per well in a 48-well plate) were cultured in Iscove’s (Corning) complete media, unless specified otherwise, with lipopolysaccharide (LPS) (20 µg/mL; InvivoGen) for up to three days. Enriched CD8+ T cells (5 x 105 cells per well) were cultured in Iscove’s complete media containing human recombinant IL-2 (100 units/mL) in 48 well plates coated with anti-CD3/anti- CD28 antibodies (1 µg/mL; Bio X Cell). For stimulation of CD8+ T cells from P14 mice, single cell suspensions from whole spleens were prepared and cultured in Iscove’s complete media,

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unless specified otherwise, containing gp33-41 peptide (1µg/mL) plus IL-2 (100 units/mL). For glucose deprivation experiments, B cells or P14 CD8+ T cells were cultured in glucose-free/2mM glutamine replete RPMI (Gibco) supplemented with Fetal Calf Serum (dialyzed overnight with SnakeSkin dialysis tubing 10K MWCO; Thermo Fisher) and where specified 10 mM D-(+)- glucose (Sigma) or 10 mM D-(+)-galactose (Sigma). The following compounds were used in cell culture experiments: mDivi-1 (Cayman), Chloroquine (Enzo), Metformin (AMPK activator; Calbiochem), Rapamycin (mTOR inhibitor; Selleckchem), N-Acetyl-L-cysteine (NAC; Sigma), 2-Deoxy-D-glucose (2-DG; Sigma), LY294002 (PI3K inhibitor; Cell Signaling), Triciribine (AKT inhibitor; Cayman), AS1842856 (FoxO1 inhibitor; Calbiochem), and Oligomycin (Sigma).

2.3 Retroviral transduction

Enriched B cells or CD8+ T cells were stimulated with LPS or plate-bound anti-

CD3/anti-CD28 antibodies plus IL-2, respectively, for 36 hours prior to retroviral transduction. Cells were then re-plated in supernatant collected prior to transduction. Retroviral constructs used were as follows: mCherry-alpha-tubulin fusion protein (Day et al., 2009); mouse dominant negative Drp-1 (K38A), a gift from David Chan (Addgene plasmid # 26049) that we

subsequently cloned into the MSCV-IRES-Thy1.1 plasmid, a gift from Anjana Rao (Addgene plasmid # 17442); and hexokinase 2, made by subcloning the rat hexokinase 2 coding sequence into an MSCV-IRES-GFP vector. Empty MSCV-IRES- Thy1.1 or MSCV-IRES-GFP vectors were used as controls. The pMIG-IRES-truncated human NGFR and its derivative exofacial- FLAG epitope tagged rat Glut1 were gifts from Dr. J. Rathmell (Wieman et al., 2007); GFP- wild-type Rab11a and GFP-dominant negative Rab11a (S25N) were made by subcloning human Rab11a (Addgene plasmid #s 12674 and 12678, respectively) (Choudhury et al., 2002) into the

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MSCV-IRES-Thy1.1 vector; and Far Red-tubulin by subcloning pmiRFP703-Tubulin (Addgene plasmid # 79991) into the MSCV-IRES-Thy1.1 vector. Empty MSCV IRES- Thy1.1 or MSCV- IRES-truncated NGFR (Addgene plasmid # 27489) vectors were used as controls.

2.4 Adoptive Transfers and Infectious Challenges

Resting CD8+ T cells from P14 mice (Thy1.1+) were purified with the CD8+ T Cell Isolation Kit (Miltenyi Biotec). 3 × 106 CD8+ T cells and adoptively transferred intravenously (i.v.) into wild- type congenic C57BL/6 mice (Thy1.2+). To generate acutely resolved systemic infections, mice were infected with 5 × 103 PFUs of Listeria monocytogenes expressing gp33-41 (LMgp33) by intravenous injection. The animals were sacrificed by day 4 post-infection for sorting of CD8+ Thy1.2- cells for immunofluorescence.

For surface Glut1 level experiments, P14 Myc-Tagged Glut1 transgenic mice were infected with 2 × 105 plaque-forming units (PFUs) of LCMV, Armstrong strain, by intraperitoneal (i.p.) injection. The animals were sacrificed by day 7 post-infection for FACS analysis.

2.5 Flow cytometry

For detection of intranuclear/intracellular antigens, cells were initially washed in PBS and stained by antibody against selected surface antigens and by LIVE/DEAD Fixable Green or Red Dead Cell Stain Kit (Thermo Fisher) and then treated with the Transcription Factor Buffer Staining Set (eBioscience) according to the manufacturer’s instructions. Antibodies specific for the following antigens were used: Pax5 (clone 1H9 conjugated to Alexa Fluor 488 or PerCP- Cy5.5 from Biolegend or APC from eBioscience; 1:200), IRF4 (clone 3E4 conjugated to PE or eFluor660 from eBioscience; 1:100), TCF1 (clone C63D9 conjugated to Alexa Fluor 647 from

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Cell Signaling; 1:100), GFP (clone 9F9.F9 conjugated to Alexa Fluor 488 from Sigma; 1:100), and Thy-1.1 (clone OX-7 conjugated to PerCP-Cy5.5 from Biolegend; 1:200). BD LSRII, Fortessa, and FACSAria II flow cytometers with FACSDiva software were used to analyze and/or purify cells. Flow cytometry data was analyzed using FlowJo software (versions 8 and 10).

2.6 Other dye staining protocols

Mitochondrial membrane potential was measured by incubating cells with MitoTracker® Red CMXRos (Cell Signaling) (100nM for flow cytometry; 200nM for confocal microscopy) for 15 to 20 minutes in the culture medium at 37 degrees Celsius. Autophagic organelles were labeled with CYTO-ID Autophagy Detection Kit (Enzo; 1:1000 dilution of stock) according to the manufacturer’s instructions. Reactive Oxygen Species (ROS) were detected by incubating cells with MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher; 5 µM) for 15 minutes at 37 °C in media or CM-H2DCFDA (Thermo Fisher; 5 µM) for 30 minutes at 37 °C in media. Glucose uptake was measured by incubating cells in 2-NBDG (Cayman; 100 µM) for 45 minutes at 37 °C in glucose-free media.

2.7 Confocal microscopy

Confocal microcopy was performed as described (J. T. Chang et al., 2011; W. H. Lin et al., 2015). Using transmitted light morphology, tubulin bridge staining, and DNA staining, specific identification of telophase or cytokinetic sibling pairs with exclusion of any spurious

neighboring-but-unrelated cell pairs is achieved (Lin et al., 2015). For fixed cell imaging, cells were adhered to poly-l-lysine (Sigma) coated coverslips (#1.5 Thermo Fisher) and treated with freshly prepared 3.7 % PFA (Sigma) diluted in cell culture media (pH 6.8) in order to preserve

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mitochondrial morphology and MitoTracker Red FM fluorescence intensity. Following brief treatment with 0.1% Triton, cells were stained with antibody against α-Tubulin (clone YOL1/34 Abcam; 1:300) and then anti-rabbit antibody conjugated to Alexa Fluor 568 (Thermo Fisher) in a PBS solution containing 0.01 % saponin and 0.25 % fish skin gelatin (Sigma). For live cell

imaging, cells were transferred to poly-l-lysine coated 8 chambered coverglass wells (#1 LabTek) in warm cell culture media and allowed to adhere briefly before imaging. Images were acquired on inverted confocal microscopes (Zeiss LSM710 or Nikon Ti Eclipse) and processed

using ImageJ (v.1.46r) or Fiji (v.2.0) software to project z stacks, apply a minimal smooth filter for some displayed images, rotate, change pseudocolors, convert to RGB, and add scale bars or time scales. Total fluorescence in defined regions of single cells was quantitated using the integrated density function in ImageJ.

2.8 Statistical Analyses

Significance between experimental groups was determined using two-tailed t tests or two-tailed ratio t tests for paired data, two-tailed Mann-Whitney test for unpaired data, or repeated-

measures one-way ANOVA. Two-tailed Fisher’s exact test was used to determine significance for contingency statistics of microscopy data comparing asymmetric versus symmetric

fluorescence intensity of molecules between sibling pairs. Statistical analyses and summary statistics to determine means and SDs were carried out using GraphPad Prism software v. 7 (_∗p < 0.05, _∗∗p < 0.01, and _∗∗∗p < 0.005).

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Chapter 3: Asymmetric mitochondrial stasis in daughter cells guides the